Insulin, a metabolic hormone, maintains the glucose homeostasis by increasing the glucose utilisation by adipose and skeletal muscle and by inhibiting the glucose production from the liver (Kahn, 1994, Diabetes 43: 1066–1084). Insulin action stimulates the glycogen synthesis in muscle by increasing the glucose uptake involving many enzymes including hexokinase. More than 80% of insulin stimulated glucose uptake takes place in skeletal muscle, the rest being by the other tissues, primarily adipose (DeFronzo et al, 1981, Diabetes 30: 1000–1007). The hyperglycaemic condition that prevails in the type II diabetic patients is due the defective muscle glycogen synthesis which in turn is due to the reduced glucose uptake as a result of insulin resistance in the skeletal muscle. So, insulin resistance in the skeletal muscle leads to higher glucose levels in the blood circulation and hence to type II diabetes. Peripheral insulin resistance in skeletal muscle is the major contributor towards the development of type II diabetes and the defects in this tissue precedes the clinical diagnosis of the disease (Vaag et al, 1992, J. Clin. Invest 89: 782–788). Reduced glucose transport into the skeletal muscle in response to insulin has been clearly established in type II diabetic patients (DeFronzo et al, 1985, J. Clin. Invest. 76: 149–155; Andreasson et al, 1991, Acta Physiol. Scand. 142: 255–260; Dohm et al, 1988, J. Clin. Invest. 82: 486–494). Identification of methods that enhance insulin action in this tissue will result in the glucose homeostasis of whole body. There are animal models to study this, use of which is limited in many ways. An in vitro cell based model will be very useful which is not available.
Insulin resistance plays a major role in the development of type II diabetes, which accounts for 90–95% of all cases of diabetes. In 1995, an estimated 135 million people were affected by diabetes, with more than half comprising from only three countries-China, India, and USA (King et al, 1998, Diabetes Care 21: 1414–1431). In 1997, nearly 16 million people in the USA had diabetes (Engelgau et. al, 2000, Diabetes Care 23: 1563–1580). It has been estimated by the year 2025 there will be approximately 300 million people affected by diabetes mellitus worldwide (King et al, 1998, Diabetes Care 21: 1414–1431). Most of the people will be from the above mentioned three countries. Another study suggests, that by the year 2020, there will be approximately 250 million people suffering from type II diabetes (O'Rahilly, 1997, BMJ 314: 955–959). Death rates due to diabetes have increased by about 30% in the past 12 years and the life expectancy of persons having diabetes is approximately 15 years less than those who do not have (Olefsky, 2001, JAMA 285: 628–632). Moreover, diabetic retinopathy is the leading cause of blindness in adults aged 20 through 74 years and the diabetic kidney disease accounts for 40% of all the new cases of end stage renal disease (Olefsky, 2001, JAMA 285: 628–632).
Insulin action is mediated through binding and activation of insulin to its receptor. Receptor consists of two α and two β subunits to form a heterotetrameric complex linked by disulphide bridges (Pessin and Saltiel, 2000, J. Clin. Invest. 106: 165–169; Kahn, 1995, Nature 373: 384–385). It is well known that tyrosine phosphorylation activates the receptor, whereas phosphorylation on serine/threonine residues hinders the activation of the receptor (Pessin and Saltiel, 2000, J. Clin. Invest. 106: 165–169). Insulin binding mediates many biological responses that could be manifested as metabolic processes (changes in carbohydrate, lipid or protein metabolism) as well as mitogenic processes (alteration in the growth, differentiation, DNA synthesis, regulation of gene expression) (Meyer and White, 1996, Annu. Rev. Pharmacol. Toxicol. 36: 615–658). The activated IR results in the tyrosine phosphorylation of many cytoplasmic substrates, including the members of IRS family (IRS1/2/3/4), the Shc adaptor protein isoforms, SIRP family members, Gab-1, Cb1, APS and dok-2 (Pessin and Saltiel, 2000, J. Clin. Invest. 106: 165–169). These docking proteins interact with the SH2 domain containing adapter proteins such as PI 3-K, Grb-2, Crk etc and SH2 domain containing enzymes such as Fyn, Csk, SHIP etc (Virkamaki et al, 1999, J. Clin. Invest. 103: 931–950; Ryder et al, 2001, Front. Biosci. 6: d154–163).
Insulin resistance is defined as the impaired biological response to either exogenous or endogenous insulin. Resistance to insulin may be manifested in several diseases, including type II diabetes, obesity, dyslipidemia, hypertension, polycystic ovarian syndrome, etc. In connection to insulin signalling in skeletal muscle, impaired IR tyrosine kinase activity has been reported from subjects with NIDDM and obesity (Nolan et al, 1994, J. Clin. Endocrinol. Metab. 78: 471–477). Similarly, defects in IR as well as insulin receptor substrate-1 (IRS-1) phosphorylation were noted in skeletal muscle of severely obese insulin-resistant subjects (Bjornholm et at, 1997, Diabetes 46: 524–527). Reduced phosphorylation of IRS-1 has been reported in skeletal muscle from NIDDM subjects after in vivo insulin infusion (Goodyear et al, 1995, J. Clin. Invest. 95: 2195–2204). Recently it has been shown that phosphorylation of IR and IRS-1 and P13-K (phosphatidyl inositol 3-kinase) activity was drastically reduced in obese non-diabetics and was virtually absent in NIDDM subjects in the skeletal muscle, but the MAPK pathway was unaffected (Cusi et al, 2000, J. Clin. Invest. 105: 311–320). In skeletal muscle IRS expression appears to be restricted to IRS-1 and IRS-2. In another recent study, results showed that IRS-1 plays a prominent role in insulin resistance in skeletal muscle (Kido et al, 2000, J. Clin. Invest. 105: 199–205). Based upon these observations, Ryder et al has stated that suppression of insulin action at the level of IR, IRS-1, or PI 3-K is likely to contribute to reduced glucose transport in the skeletal muscle from diabetic patients (Ryder et al, 2001, Front. Biosci. 6: d154–163).
Thiazolidinediones (TZDs) are a new class of anti-diabetic agents and include three compounds that have been allowed to use clinically: troglitazone, pioglitazone and rosiglitazone (Kahn et al, 2000, J. Clin. Invest. 106:1305–1307). However, troglitazone was banned recently in few countries due to its hepatotoxicity. TZDs has been shown to improve the insulin action in vivo (Olefsky, 2000, J. Clin. Invest. 106: 467–472; Hayakawa, 1996, Biochem. Biophys. Res. Commun. 223:439–444). Therefore, atleast pioglitazone, and/or rosiglitazone can very well be used to validate any method of insulin sensitisation.
Screening can be of two types: it may involve the screening of the non-symptomatic mass having type II diabetes to assess the health care burden on the nation or it may involve the screening of the anti-diabetic agents (Engelgau et al, 2000, Diabetes Care 23: 1563–1580). Screening of non-symptomatic mass involves questionnaire and biochemical tests. Questionnaire is very popular, and inexpensive, but the performance in regard to the assessment of the disease is poor. However, it is very good for educating the people as well as making them aware (Engelgau et al, 2000, Diabetes Care 23: 1563–1580). Biochemical tests are based upon the glucose measurement in the blood making these tests more reliable. However, the cut off point where the people will be considered glucose intolerant or diabetic are not well defined (Engelgau et al, 2000, Diabetes Care 23: 1563–1580). As far as the screening for anti-diabetic agents is concerned, there are few animal models available. However, screening large number of compounds would be very expensive. Moreover, the variability among the animals makes it harder to compare the results and it also has the potential problems of animal ethics. So an in vitro model in skeletal muscle will be highly relevant. But there is not a single model available based on one of the most important tissues like muscle in regard to the screening for the diabetic people.
So, altogether, skeletal muscle is one of the most important tissues, affected severely due to insulin resistance. There are few animal models available, but there use is limited. To screen the non-symptomatic population periodically as recommended by WHO (World Health Organization), BDA (British Diabetic Association), ADA (American Diabetic Association), ACP (American College of Physicians), AAFP (American Academy of Family Physicians) to reduce the health care burden of a nation (Engelgau et al, 2000, Diabetes Care 23: 1563–1580), and to screen the anti-diabetic compounds targeted against insulin resistance, there is no in vitro insulin resistant skeletal muscle based protocol and model available.
Reference may be made to Conejo and Lorenzo, 2001, J. Cell Physiol. 187: 96–108; Conejo et al, 2001, J. Cell Physiol. 186: 82–94 wherein C2C12 cells has been differentiated in serum free medium in the presence of insulin. It was shown that insulin simulation leads to the activation of extracellular signal-regulated kinase (ERK) and p38 MAPK (mitogen-activated protein kinase). However, the protocol of this study was to elucidate the basic mechanism of insulin signalling and not to generate insulin resistance.
The continues presence of insulin would be simulation of the physiological condition, which is supposed to desensitise IR and associated signal transduction pathway in insulin resistance, as reported in the literature Ricort et al. (1995, Diabetologia 38: 1148–1156).
Thomson et al., (J. Biol. Chem., 1997, 272: 779–784) has shown that chronic insulin treatment of 3T3-L1 adipocytes results in reduced glucose transport. Further stimulation with insulin does not alter the condition. Therefore, insulin non-responsiveness due to chronic insulin treatment is known in literature. The Applicants are not trying to patent this concept, the Applicants wish to patent skeletal muscle model, culturing of which is different than adipose tissue, that has resulted into resistance to insulin in skeletal muscle.
Ricort et al, (1995, Diabetologia 38: 1148–1156) have shown that chronic treatment of 3T3-L1 adipocytes with insulin results in the reduced tyrosine phosphorylation of IR and IRS-1 and does not respond to further stimulation by insulin. After conforming the correctness of differentiated skeletal muscle cells, the Applicants have tested whether the differentiated muscle have generated insulin non-responsiveness, as measured by decrease in its IR and IRS-1 tyrosine phosphorylation. The results showed that tyrosine phosphorylation of IR and IRS-1 was indeed reduced. This is a validation, based on literature, of the development of insulin resistance under the subjected conditions. This is the first in vitro model in skeletal muscle, where the insulin resistance has been developed and validated, based on literature, a through the proximal steps of insulin signalling.
It has been shown that pioglitazone enhances the tyrosine phosphorylation of IR and IRS-1 in the insulin resistant Wistar fatty rats in the skeletal muscles (Hayakawa et al., BBRC, 1996, 223: 439–444). This implies that pioglitazone is a drug which can enhance the tyrosine phosphorylation of IR and IRS-1 in skeletal muscle. To validate this model further, in connection to whether a known sensitizer like pioglitazone can sensitize (i.e., increase in tyrosine phosphorylation of IR or IRS-1 of the insulin non responsive cells due to further treatment of insulin in the presence of pioglitazone) the insulin non-responsiveness of the skeletal muscle cells that the Applicants generated, and, whether this model will be useful for screening unknown compounds against insulin resistance, the Applicants tested the effect of pioglitazone on the insulin non-responsiveness skeletal muscle cells and measured IR and IRS-1 tyrosine phosphorylation. Data thus obtained shows that tyrosine phosphorylation of IR and IRS-1 was increased to normal level, essentially meaning, that the insulin non-responsiveness skeletal muscle cell culture model can be sensitised, thereby it is valid. Therefore, the Applicants have recorded information from literature (like in any other patent) but developed a unique and extremely useful model in skeletal muscle, supported by appropriate validation regarding its sensitisation and its prospect of use in screening new chemical entities against diabetes associated with insulin resistance.
The main disadvantages with the insulin non-responsive animal models available are as follows:    a. requires unacceptable number of animals to screen thousands of compounds being generated world-wide;    b. cost associated with the number of animals required, is prohibitory;    c. severe limitations due to the regulatory laws associated with animal ethics, and    d. variations between animal to animal lead to unreliability of data. To obtain statistically significant data, large number of animals are required to be experimented upon resulting into falling into the aforesaid limitations.